1. Field of the Invention
This invention relates to isolating a DNA sample from a heterogeneous mixture of DNA and other compounds. Specifically, the invention relates to isolating a DNA sample without the use of hazardous or toxic chemicals or time-consuming physical separation techniques. The invention relates in particular to isolating a plasmid DNA sample from, for example, a cleared bacterial lysate. The invention provides an apparatus and methods for using the apparatus to rapidly and economically isolate a DNA sample from such mixtures.
2. Description of the Prior Art
Modem molecular biology requires the isolation of DNA from a variety of sources comprising mixtures of the DNA with heterologous material such as proteins, lipids and other cellular constituents. Particularly important examples of such heterogeneous mixtures include cleared bacterial or yeast lysates containing plasmid or cosmid DNA, recombinant phage lysates, polymerase chain reaction mixtures, and other reaction mixtures employed in recombinant DNA methodologies. Conventional methods for separating DNA from such contaminants include extraction with a variety of organic solvents, including phenol, chloroform, and diethyl ether, followed by precipitation with ethanol, and equilibrium density centrifugation using gradients of cesium chloride, sodium chloride or sucrose. These conventional methods suffer from the disadvantages of being time-consuming and laborious, and they also involve the use of chemical reagents that are hazardous and/or toxic to the worker, the environment, or both.
Recently, it has been recognized that DNA preferentially and reversibly can be caused to bind to glass or other silicate, preferably ground glass beads, and that such bound DNA can be eluted from glass using low ionic strength buffers. This DNA binding method has lead to the of a number of commercial DNA isolation kits of a prepared slurry of glass beads and associated solutions. While advantageous for providing a commercially-available embodiment of glass-based DNA binding methods, these kits can be improved in several ways.
First, and most importantly, in the use of these kits DNA must be pelleted with the beads from solution, washed and then eluted from the beads. This mode of preparation, understood in the art as a batch mode of preparation, is disadvantageous because no provision is made for physically separating the glass beads from the eluted DNA. As a result, in the practice of these methods, it is difficult to completely remove all of the glass beads from the eluted DNA. A mixture of glass beads and a DNA sample in the final elution can compromise the accuracy of assessment of the DNA concentration and purity in the sample. The beads will continue to bind and sequester the DNA on storage, and may bind the majority of the DNA in the sample upon prolonged incubation, rendering the DNA unusable.
A second limitation of glass bead binding-based DNA isolation methods is that the glass bead binding method is a single-step procedure. It will be understood that the purity of a DNA sample, as assayed by the elimination of contaminating species such as proteins, is necessarily compromised in any single-step isolation procedure. Such single-step DNA isolation methods are known in the art, for example, methods employing the Wizard miniprep system (commercially available from Promega Corp., Madison, Wis.) and the Prep-a-Gene system (commercially available from BioRad, Inc., Hercules, Calif.). Each of these DNA isolation systems employs a single, silica-based resin in a single-step DNA purification scheme. In practice, these systems do not provide DNA having the purity required for many molecular biological procedures. Additionally, the reliability of these systems is poor in providing high-purity DNA for certain applications (e.g., fluorescent label-dependent DNA sequencing).
European Patent Application Publication No. 0263934A1, published 20 Apr. 1988 discloses the use of porous silica gel modified to have affinity ligands covalently bound to the surface of the gel particles, for DNA purification.
International Patent Application Publication No. WO93/11218, published 10 Jun. 1993, discloses methods of DNA purification comprising passing the DNA sample through a filter, the filter being characterized as having decreasing pore size in the direction of sample flow.
Another methodology used in the DNA isolation arts employs ion-exchange resins that bind to DNA, the DNA being differentially eluted from such resins using salts or other ionic competitors. Since DNA is negatively charged at all relevant pH values due to the presence of a phosphate residue between each nucleotide, it was recognized that anion exchange columns would be particularly useful for DNA sample isolations. However, anion exchange resin-based methods suffer from the drawback that anionic proteins are also bound by such anion exchange resins, and separation of DNA from such proteins is frequently impossible. In addition, elution of the DNA from such resins involves washing the resin with a salt solution, thereby requiring the eluted DNA to be purified from the salt solution by, e.g., ethanol precipitation. Ethanol precipitation can lead to loss of sample, particularly DNA samples containing small amounts of DNA or at low concentrations, and can also lead to excess salt, ethanol, or both being present in the final DNA sample. These contaminants are undesirable because they interfere with, inter alia, enzymatic manipulation of the DNA sample.
U.S. Pat. No. 5,057,426, issued Oct. 15, 1991, discloses a method for purifying a DNA sample comprising passing the DNA sample through a porous matrix that is a modified silica gel that is an anion exchange resin.
European Patent Specification, Publication No. 0268946B1, issued 15 Sep. 1993, application published 01 Jun. 1988, discloses a method for purifying a DNA sample comprising passing the DNA sample through a porous matrix that is a modified silica gel that is an anion exchange resin.
International Patent Application, Publication No. WO92/07863, published 14 May 1992, discloses a method for isolating DNA from a cell sample containing the DNA, comprising lysing the cells containing the DNA on a porous matrix, wherein the DNA is fixed on the surface of the porous matrix and then differentially eluted therefrom.
International Patent Application, Publication No. WO88/09201, published 1 Dec. 1988, discloses a device, adapted for use with a pipette, for purifying DNA from a sample.
Experimental protocols whereby these DNA isolation methods are combined are known in the prior art, but they have proven time-, labor- and material-intensive due to the perceived need to perform each DNA binding and elution step independently. An example of such a prior art recognized protocol is embodied in the Qiawell-Plus kit (commercially available from Qiagen Corp., Chatsworth, N.C.). This procedure requires two separate columns: one containing an anion exchange resin, and the other containing a silica resin. In the use of these columns, a DNA sample is first bound to the anion exchange column, washed repeatedly, then eluted from the column and the eluate recovered. The DNA sample must then be bound to the second column, washed repeatedly, and then eluted and recovered from the second column. This protocol involves disadvantageous mounts of manual manipulation of reagents, columns and DNA samples, and typically requires almost an hour of worker handling and processing to purify a DNA sample. As a result, in practice this protocol achieves only suboptimal throughput efficiencies and is time-, materials- and labor-intensive.
International Patent Application, Publication No. WO93/11221, published 10 Jun. 1993, discloses a method and apparatus for purifying nucleic acids, specifically plasmids and genomic DNA from cells, the method comprising treating the nucleic acid-containing solutions with an anion exchange resin in a low ionic strength buffer; desorbing the DNA from the anion exchange resin using a higher ionic strength buffer; treating the desorbed DNA with a mineral base in the presence of a higher ionic strength buffer or with lower alcohols/polyglycols with adsorption of the DNA onto the mineral base; and desorbing the DNA from the mineral base with water or a low ionic strength buffer.
International Patent Application, Publication No. WO93/11218, published 10 Jun. 1993, discloses methods of DNA purification comprising passing the DNA sample through a filter, the filter being characterized as having decreasing pore size in the direction of sample flow.
There is a need in the DNA isolation arts for a rapid, sensitive, and economical method of isolating DNA from a heterogeneous mixture. In particular, there is a need for combining the techniques of ion exchange chromatography and glass bead DNA binding to provide a fast, simple method of specifically isolating a DNA sample from a mixture that does not involve separation of these methodologies into two separate, unrelated, and time-consuming steps of a complicated isolation procedure.